Design and development of a magnetic field-enabled platform for delivering polymer-coated iron oxide nanoparticles to breast cancer cells

The current literature mostly contains relatively vague descriptions of techniques for implementing in vitro magnetic targeting delivery of iron oxide nanoparticles (IONPs), leading to irreproducible processes and incomparable findings. This discrepancy often arises from the varying exposure of IONPs to the non-uniform magnetic field and differences in the concentration of the polymer-coated IONPs. Hence, we meticulously designed and built a system comprising a platform constructed from polyoxymethylene sheets, which securely holds the permanent magnets, and the cell culture plate. We also tailored the preparation process of the IONPs and the in vitro toxicity studies. The inherent characteristics of IONPs are further enhanced by their coating with natural polymers, alginate (Alg) and chitosan (CS).• The design and construction of the platform were carried out using a laser engraving/cutting machine along with graphic design software. The precise locations of the permanent magnets relative to the cell culture plate were determined via a Gaussmeter.• The quantities of the components in the formulation and the method for fabricating the CS/Alg-coated IONPs (CS/Alg-IONPs) were optimized to ensure that the desired physicochemical properties were obtained.• The cultivation and cytotoxicity evaluation of the fabricated CS/Alg-IONPs against MCF-7 breast cancer cells were described.


a b s t r a c t
The current literature mostly contains relatively vague descriptions of techniques for implementing in vitro magnetic targeting delivery of iron oxide nanoparticles (IONPs), leading to irreproducible processes and incomparable findings. This discrepancy often arises from the varying exposure of IONPs to the non-uniform magnetic field and differences in the concentration of the polymer-coated IONPs. Hence, we meticulously designed and built a system comprising a platform constructed from polyoxymethylene sheets, which securely holds the permanent magnets, and the cell culture plate. We also tailored the preparation process of the IONPs and the in vitro toxicity studies. The inherent characteristics of IONPs are further enhanced by their coating with natural polymers, alginate (Alg) and chitosan (CS).
• The design and construction of the platform were carried out using a laser engraving/cutting machine along with graphic design software. The precise locations of the permanent magnets relative to the cell culture plate were determined via a Gaussmeter. • The quantities of the components in the formulation and the method for fabricating the CS/Alg-coated IONPs (CS/Alg-IONPs) were optimized to ensure that the desired physicochemical properties were obtained. • The cultivation and cytotoxicity evaluation of the fabricated CS/Alg-IONPs against MCF-7 breast cancer cells were described. Specifications

Method details
Construction of the platform for the permanent magnets and 96-well plates

Materials
Note : The materials are listed in the sequence in which they appear in the procedure. Note: POM sheets are selected as construction materials for our platform designed to anchor the permanent magnets and 96well plate. POM sheets are advantageous for laser cutting since they neither emit acids nor toxic fumes. Their excellent dimensional stability during high-precision cutting and glossy surface finish make them ideal for our purposes. Furthermore, their high resistance to chemicals and melting point of 183 °C render them suitable for sterilization through autoclaving. The platform, comprising three interconnected layers via machine screws, ensures optimal stability and manageability, with each layer being 5 mm thick. a. Craft sketches for each component of the platform utilizing the graphic design software CorelDRAW.
Note: If desired, an image file (JPG, PNG, or GIF) can be directly imported into the graphic design space of CorelDRAW as an alternative. b. Select the dimensions for each component: (1) Top ( Fig. 1 A) • Engineered to snugly fit the lower body of the 96-well plate.
• The design's thickness ensures sufficient clearance to avoid direct contact with the lid of the 96-well plate, thus eliminating potential contamination. (2) Middle ( Fig. 1 B) • Designed to secure the permanent magnets.
• Each compartment has a 5.5 mm diameter to accommodate each 5 mm permanent magnet.
• The permanent magnet's 5 mm diameter, smaller than the well plate's bottom diameter (6.21 mm), compensates for the magnetic field strength of the magnet, which is most potent at its center and edges. This also provides a clear field of view for cell examination under the influence of magnets. (3) Bottom ( Fig. 1 C) • Works in conjunction with the middle layer to affix the magnet to its surface and maintain overall platform stability. c. Translate or create the design images into a vector format.
Note : Vector images can be enlarged without compromising the quality of the original image's lines and colors. A vector path with minimal line thickness is required by the laser machine.   c. Define the file type that the program will interpret: • Cutting data: PLT-HP-GL/2 Plotter file • Cutting area: Current page d. Select the "Cutting " mode of laser operation and adjust the parameters as listed below: • Speed = rate of laser shooting (5 mm/s) • Power = intensity of the laser (50 Watts) • Frame = shape of the frame (Rectangle) • Refer = relative position of the laser head (TopRight) • Refer-X = x coordinate of the laser head • Refer-Y = y coordinate of the laser head e. Manually set the distance between the POM plate and the laser head to approximately 8 mm. f. Click "Starting " to initiate the laser cutting process.
Note : Prior to activating the laser operation, ensure that the air pump is turned on to avert burning the material due to generated heat. A few attempts may be necessary to find the optimal settings in the software for the material. Only change one operational condition at a time. For power, begin from a low value and gradually increase until the appropriate conditions are met. g. Use a caliper to verify if the POM plate has the correct dimensions.

Measurement of the magnetic field (B) of the permanent magnets on the empty 96-well plate using a Gaussmeter
Note : Lake Shore Cryotronics, Inc. has discontinued the supply of Model 455-DSP Gaussmeter and recommends using Model F71 instead. Small permanent magnets are typically used for biomedical applications and are classified as low field gradients [1] . A visual depiction of the platform assembly and the magnetic field measurement is shown in Fig. 2 . a. Allow the Gaussmeter and Hall probe to equilibrate at least 30 min before use. b. Attach the 96-well plate to the top POM sheet. c. Stack the middle POM sheet on top of the base POM sheet. d. Place three permanent magnets (total length, 9 mm) in each compartment (letters F to H) of the middle POM sheet and secure them in place.
Note : A process of trial and error is used to determine the positions of the permanent magnets on the 96-well plate. Compartments labeled A to C are used for experiments without permanent magnets. Compartments D and E are left vacant because positioning permanent magnets in these compartments results in magnetic field readings in compartments B and C, even without magnets present in these locations. Leaving compartments D and E empty ensures a reliable comparison of the nanoparticle (NP) cytotoxicity results between compartments A to C (cells treated in the absence of permanent magnets) and compartments F to H (cells treated in the presence of the permanent magnets). e. Place the top layer (96-well plate + top POM sheet) over the stacked assembly (middle POM sheet with magnets + base POM sheet). f. Carefully lift the entire assembly and secure all plates with the screws. g. Place the fixed assembly onto a clean and uncluttered working surface. h. Gently remove the lid of the 96-well plate. i. Measure the B of each magnet by vertically positioning the Hall probe and attaching the round axial sensor to the bottom of the 96-well plate. Note : Maintaining the Hall probe perpendicular to the assembly's resting plane ensures a maximum reading output from the Gaussmeter (zero percent reading error). Additionally, a vertical distance (1.2 mm) exists between the internal well bottom (where cells attach) and the external well bottom (where the magnet directly contacts). Hence, B is not measured directly on the surface of the permanent magnet. The well bottom (diameter, 6.35 mm) suits the attachment of the Hall probe (diameter, 6.20 mm). This approach offers a realistic approximation of the actual B experienced by the NP-treated cells. The mean (standard deviation) B of the magnets used for the 96-well plates is 0.3149 (2.53) Tesla ( n = 6).

Materials
Note : The materials are listed in the sequence in which they appear in the procedure.

Preparation of the chitosan (CS) solution
Prepare a 0.23 mg/mL CS solution with a total volume of 50 mL as follows: Note : This volume is adequate for preparing four formulations.
a. Prepare 100 mL of a 1% ( v/v ) acetic acid solution by diluting 1 mL of glacial acetic acid with 95 mL of ultrapure water. Add ultrapure water to achieve a total volume of 100 mL. b. Pour 40 mL of the 1% ( v/v ) acetic acid solution into a 250 mL Erlenmeyer flask. c. Weigh out 11.5 mg of CS powder and gradually add it to the acetic acid solution. d. Cover the flask's opening with Parafilm R ○ M and shake at 150 rpm for 24 h to ensure total dissolution of CS.
e. Adjust the pH of the CS solution to 5.0 using 2 M NaOH. Then, add ultrapure water to make a total volume of 50 mL. Note: Dissolve 0.8 g of NaOH pellets in ultrapure water to make 10 mL of 2 M NaOH. Let the solution cool to room temperature before use. f. Set up the filtration apparatus: Fit the filter adapter onto the opening of a 500 mL filtering flask. Secure the Buchner filter funnel on top of the adapter, then position the membrane filter at the center of the funnel. g. Moisten the membrane filter and the funnel's interior with a small portion of the solution. h. Carefully pour the solution against the sides of the funnel, avoiding direct contact with the membrane filter until the funnel is two-thirds full. i. Turn the vacuum on, ensuring the membrane filter does not dry until all the solution is filtered. j. Transfer the filtered solution to a 500 mL bottle and tightly close it. k. Store the solution at 4 °C for up to 3 days.

Preparation of the solutions of FeCl 2 ·4H 2 O and FeCl 3 ·6H 2 O
Freshly prepare 50 mL of each c. Seal each tube, vortex mix for 1 min, and sonicate the tubes at 9 W for 10 min while keeping the bath cool with ice. d. Add 20 mL of ultrapure water to each tube, then vortex mix again. Note : Alternatively, invert the tubes 20 times for adequate mixing since blending a 50 mL solution in a 50 mL conical tube may need some pressure adjustment for efficient vortex mixer use. Use these solutions within 8 h.

Preparation of the CS/Alg-IONPs
The procedure for synthesizing CS/Alg-IONPs is adapted from previous research [2] . The quantities of Alg, CS, FeCl 2 ·4H 2 O, and FeCl 3 ·6H 2 O have been optimized to obtain desirable characteristics of the polymer-coated IONPs. Note : Set the syringe pump at a flow rate of 40 mL/h, with a syringe diameter of 20.12 mm, and a volume of 10 mL. Nitrogen gas is used to saturate the mixing vessels at all reaction stages. Mixing is performed at 1000 rpm at room temperature. All solutions should also be at room temperature when used. j. Place the beaker on the permanent magnet (50 × 50 × 20 mm 3 ) to separate the Alg-IONPs from the dispersion. Note : During this step, the first decantation process will remove excess ammonia, which has a powerful and irritating odor. Wear goggles, nitrile gloves, and a face mask for protection. Additionally, keep the permanent magnet away from magnetic materials or metals that could be attracted to it. k. Add 80 mL of ultrapure water to the Alg-IONPs, gently swirl to mix, and separate the Alg-IONPs by placing the beaker on the permanent magnet. Note : Let the Alg-IONPs settle for 2-3 min before discarding the supernatant. Repeat this process until the washings reach a pH of approximately 7.0. Also, check the washings for chloride ions using 1% ( w/v ) AgNO 3 . Four washing attempts are enough to achieve a neutral pH and ensure the absence of chloride ions in the dispersion. l. Disperse the Alg-IONPs in ultrapure water to create a 50 mL dispersion, then place the magnetic bar in the mixture and cover the beaker's opening with Parafilm R ○ M secured by rubber bands.
m. Mix the dispersion for 10 min, sonicate for 20 min, and then mix for another 10 min. Note : The pH of the dispersion ranges from 6.0 to 6.5, which keeps the carboxylate groups of Alg ionized in the mixture. n. Connect the nitrogen gas-filled balloon to the beaker to saturate it with nitrogen gas. o. Fill the 20 mL syringe with 13 mL of CS solution, then deliver 10 mL of the CS solution to the dispersion using the syringe pump with the same conditions and techniques. Note : The pH of the dispersion before the addition of CS should range from 6.0 to 6.5. After adding CS, the pH of the dispersion now ranges from 5.4 to 5.7. p. Use the permanent magnet to separate the resulting CS/Alg-IONPs by attracting them to the beaker wall for 2 min. Then, carefully decant about 30 mL of the liquid from the solid NPs to remove the excess CS. q. Pour the remaining mixture into a preweighed 50 mL conical tube, and collect the CS/Alg-IONPs by attracting them to the outside of the tube wall using a permanent magnet for 2 min. Afterward, carefully decant the liquid. r. Freeze the CS/Alg-IONPs at -20 °C for 4 h and lyophilize at -80 °C for 24 h to obtain a dry powder. s. Subtract the mass of the empty conical tube from the CS/Alg-IONP-filled tube to get the mass of the CS/Alg-IONPs.
Note : A total of 50 to 55 mg of dry CS/Alg-IONP powder can be produced using this protocol.

Materials
Note : The materials are listed in the sequence in which they appear in the procedure.

Treatment of the MCF-7 cells with the CS/Alg-IONP suspension
Note : The cells should be treated with the samples after 24 h of seeding. All materials (POM plates, screws, and magnets) should be sterilized with 70% ethanol and exposed to UV light for 30 min before assembling the platform and cell-seeded 96-well plate. Assemble the materials within the BSC.
a. Assemble the 96-well plate seeded with MCF-7 cells and the platform as demonstrated in Fig. 2 A-E. b. Place the assembled structure on a clean, uncluttered work surface within the BSC. c. Remove the lid from the 96-well plate and remove the old medium from each well. d. Add 0.2 mL of the working suspension to each well.
Note : Before adding the working suspension to each well, perform the reverse and forward pipetting techniques to mix the CS/Alg-IONP suspension twice. e. Gently lift the entire assembly and incubate the cells for 1 h at 37 °C in a humidified atmosphere with 5% CO 2 by placing the assembly inside the incubator. f. After 1 h, disassemble the platform from the 96-well plate within the BSC and continue incubating the cells (in the 96-well plate) for the next 23 h. Note : To compare the effect of incubation time, the cells can also be incubated in the presence of permanent magnets for 2, 4, or 6 h.

MTT assay
Refer to a previous publication [3] for the MTT assay protocol. This custom method is designed specifically for handling IONPs.
a. After incubating the treated cells for 24 h, remove the medium, and rinse the cells twice with 0.2 mL of PBS. Note : The control (cells treated with the serum-free DMEM) should also be rinsed twice with PBS to account for the effect of rinsing on cell attachment. For each rinsing step, the PBS wash should be immediately pipetted out to prevent the settling of nanoparticles that may affect absorbance readings. b. Prepare a 0.5 mg/mL MTT solution by adding 0.7 mL of MTT stock solution to 6.3 mL of serum-free DMEM in a 15 mL conical tube, creating a 7 mL MTT solution. Vortex mix the solution. Protect the solution from light. c. Add 0.1 mL of the MTT solution to each well and incubate the cells at 37 °C for 2 h. d. Remove the MTT medium. e. Add 0.1 mL of DMSO to each well and gently shake the plate to dissolve the formazan crystals. f. Measure the optical density at 570 nm using a microplate reader. g. Calculate the cell viability (%) using: Cell viability ( % ) = N t N c × 100 where N t and N c represent the optical densities of the treated and untreated (control) cells, respectively.

Declaration of generative AI and AI-assisted technologies in the writing process
During the preparation of this work, the authors used ChatGPT 4.0 in order to improve language and readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Data availability
Data will be made available on request.